Name: macrophage differentiation and polarization into an m2-like phenotype using a human monocyte-like thp-1 leukemia cell line
Uploaded: 2021-08-02T13:00:00
Description: Watch this Scientific Journal Video about Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line at JoVE.com

The Price Institute of Surgical Research, University of Louisville, is financially supported by the John W. Price and Barbara Thruston Atwood Price Trust. The funding sources had no role in the design and conduct of the study as well as in the collection, management, analysis, and interpretation of the data.

NOTE: An overview of the steps described in this protocol is shown in Figure 1. The human monocyte-like leukemia cell line THP-1 was purchased. Short tandem repeat analysis was performed to authenticate the THP-1 cell line. Perform all steps under sterile conditions. The THP-1 monocytic cell line grows in suspension and does not attach to cell culture surfaces. Adherence can be induced by differentiating monocytes into macrophage-like cells through, e.g., mechanical stress or specific treatm...

<ol>
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J., Malik-Kale, P., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phorbol+12-myristate-13-acetate+differentiation+protocol+is+critical+to+the+interaction+of+THP-1+macrophages+with+Salmonella+Typhimurium.">The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium.</a> PLoS One. 13 (3), 0193601 (2018).</li><li>Lund, M. E., To, J., O'Brien, B. A., Donnelly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choice+of+phorbol+12-myristate+13-acetate+differentiation+protocol+influences+the+response+of+THP-1+macrophages+to+a+pro-inflammatory+stimulus.">The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.</a> Journal of Immunological Methods. 430, 64-70 (2016).</li><li>Yin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6/STAT3+pathway+intermediates+M1/M2+macrophage+polarization+during+the+development+of+hepatocellular+carcinoma.">IL-6/STAT3 pathway intermediates M1/M2 macrophage polarization during the development of hepatocellular carcinoma.</a> Journal of Cellular Biochemistry. 119 (11), 9419-9432 (2018).</li><li>O'Neill, L. A. 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B., Wittig, B., Cignarella, A., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+PMA+enhances+the+responsiveness+of+transfected+THP-1+macrophages+to+polarizing+stimuli.">Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli.</a> Journal of Immunological Methods. 402 (1-2), 76-81 (2014).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K., Dockrell, D. 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This protocol on differentiating and polarizing THP-1 monocyte-like cells within 14 days provides a method to obtain macrophages with a distinct M2-like phenotype due to long treatment incubation of cells with adequate resting periods between steps.
Certain steps are critical to this protocol. The doubling time of THP-1 monocytes is approximately 26 h. Cells can be split at a cell density of 9 x 105/mL and should be seeded at a density of 3 x 105/mL during every split. Th...

Tumor-associated macrophages (TAM) and their role in chronic inflammation, the onset of cancer, and tumor development are important targets in recent research1,2. Peripheral blood monocytes that are recruited to the tissue microenvironment of the developing tumor differentiate into macrophages and can be polarized into two main subtypes of macrophages3. The classically activated macrophage represents the primarily pro-inflammatory M1-like phenotype and the alternatively activated M2-like subtype shows predominantly anti-inflammatory characteristics4. Macrophages can switch dynamically between these two main phenotypes depending upon their cellular metabolism, with intermediate subtypes having both inflammatory and anti-inflammatory attributes5. TAM represents a heterogeneous population of both phenotypes. A tumor-promoting function and poor prognosis in different types of cancers is, however, particularly associated with M2-like macrophages6,7,8.
The functional profiles of macrophages and their interaction with other cells within the tumor microenvironment are complex and challenging to capture in a continuously changing environment during ongoing tumor development. Cell lines can provide a homogenous cell population with stable viability in culture, which can facilitate the process of demonstrating defined cellular and intercellular mechanisms. The monocyte-like THP-1 cell line is a legitimate model system for primary human monocytes9. This spontaneously immortalized cell line has been obtained from the peripheral blood of a one-year-old infant with acute monocytic leukemia9,10. The differentiation and polarization of THP-1 cells have been reported by several studies and have been performed in multiple different ways11,12,13,14. Activation and, therefore, the polarization of macrophages into an M1-like phenotype is followed by a compensatory anti-inflammatory rebound mechanism, promoting an M2-like phenotype through cytokines produced by inflammatory macrophages, such as interleukin 6 (IL-6) or itaconate15,16. This might serve as a break mechanism to attenuate an overshooting inflammatory response following cell activation17. The process of differentiating and polarizing monocytes and THP-1 monocyte-like cells into an anti-inflammatory M2-like phenotype is itself also accompanied by pro-inflammatory stimuli that must be overcome. An inflammatory cytokine response can be caused by mechanical stress18, such as changing media to refeed the cells, or adding chemical compounds to differentiate THP-1 cells, such as phorbol&#160;12-myristate 13-acetate (PMA), and induce production of tumor necrosis factor &#945; (TNF&#945;), interleukin 1&#946; (IL-1&#946;) or IL-619. This altered cytokine expression profile as a response to PMA can affect and prevent subsequent macrophage polarization20. Adequate resting periods, as reported before after PMA treatment, allow these inflammatory responses to decrease and facilitate cell polarization into a distinct M2-like phenotype21.
This protocol demonstrates a method to differentiate and polarize THP-1 monocyte-like cells into an M2-like phenotype of macrophages within 14 days.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% trypan blue</td>
			<td>VWR, Radnor, USA</td>
			<td>152-5061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1111-2821</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL&#160; Centrifuge tube</td>
			<td>VWR, Radnor, USA</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-900</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-896</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>VWR, Radnor, USA</td>
			<td>89039-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase solution 500 mL</td>
			<td>Sigma, St. Louis, USA</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100x), stabilized</td>
			<td>Sigma, St. Louis, USA</td>
			<td>A5955-100 mL</td>
			<td>with 10,000 units penicillin, 10 mg of streptomycin and 25 &#956;g of amphotericin B per mL, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Binder CO2 Incubator</td>
			<td>VWR, Radnor, USA</td>
			<td>C170-ULE3</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoOne T-75cm flask with filter cap</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>CC7682-4875</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma, St. Louis, USA</td>
			<td>D8537-500 mL</td>
			<td>PBS without calcium chloride and magnesium chloride should be used, since both can alter macrophage polarization</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5804 R (refrigerated)</td>
			<td>Eppendorf, Enfield, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol (70%)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCalibur flow cytometer</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>-</td>
			<td>The flow cytometer operates with CellQuest software (BD Biosciences)</td>
		</tr>
		<tr>
			<td>Falcon 24-well plate</td>
			<td>VWR, Radnor, USA</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD14</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555397</td>
			<td>Flow cytometry, myeloid cell marker (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD80</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>557226</td>
			<td>Flow cytometry, M1 marker (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1 &#954; Isotype Control</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>555748</td>
			<td>Flow cytometry, isotype control for CD80 (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse IgG2a, &#954; Isotype Control</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>553456</td>
			<td>Flow cytometry, isotype control for CD14 (100 tests)</td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>564220</td>
			<td>Flow cytometry, Fc block (0.25 mg)</td>
		</tr>
		<tr>
			<td>Human interleukin 13 (IL-13)</td>
			<td>R&#38;D, Minneapolis, USA</td>
			<td>IL-771-10 &#956;g</td>
			<td></td>
		</tr>
		<tr>
			<td>Human interleukin 4 (IL-4)</td>
			<td>R&#38;D, Minneapolis, USA</td>
			<td>SRP3093-20 &#956;g</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Biosafety Cabinet (Delta Series 36212/36213)</td>
			<td>Labconco, Kansas City, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution, 200 mM</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2214</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from E. coli 0111:B4</td>
			<td>Sigma, St. Louis, USA</td>
			<td>L2630-100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cell Scrapers</td>
			<td>Biotium, Fremont, USA</td>
			<td>22003</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer hemocytometer</td>
			<td>Fisher Scientific, Waltham, USA</td>
			<td>02-671-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse inverted microscope TS100</td>
			<td>Nikon, Melville, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Invitrogen, Carlsbad, USA</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Light Microscope RH-2</td>
			<td>Microscope Central, Feasterville, USA</td>
			<td>40888</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 variable pipet- Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76180-014</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 variable pipet-Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76177-990</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 variable pipet- Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76177-988</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD11b</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555388</td>
			<td>Flow cytometry, myeloid cell marker (100 tests)</td>
		</tr>
		<tr>
			<td>PE Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555749</td>
			<td>Flow cytometry, isotype control for CD11b (100 tests)</td>
		</tr>
		<tr>
			<td>PE-Cy 5 Mouse Anti-Human CD206</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>551136</td>
			<td>Flow cytometry, M2 marker (100 tests)</td>
		</tr>
		<tr>
			<td>PE-Cy 5 Mouse IgG1 &#954; Isotype Control</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>555750</td>
			<td>Flow cytometry, isotype control for CD206 (100 tests)</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, St. Louis, USA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerpette Plus pipettor</td>
			<td>VWR, Radnor, USA</td>
			<td>75856-448</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Water bath (model 183)</td>
			<td>Precision Scientific, Chicago, USA</td>
			<td>66551</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line, American Type Culture Collection (ATCC)</td>
			<td>ATCC, Manassas, USA</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
	</tbody>
</table>

macrophage differentiation and polarization into an m2-like phenotype using a human monocyte-like thp-1 leukemia cell line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like macrophages were characterized, and M2-polarization was validated using flow cytometry for Cluster of Differentiation markers (CD) CD14, CD11b, CD80 (M1-like marker), and CD206 (M2-like marker). Flow cytometry staining was performed according to the manufacturer&#39;s instructions. Macrophages were washed with PBS/5% FBS and incubated with Fc&#947;-receptor block to avoid unspecific binding. Cells were then stained with FITC-conjugated mouse anti-human CD14 and CD80 antibodies, with PE-conjugated mouse anti-human...

Watch this Scientific Journal Video about Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line at JoVE.com

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

Anti-inflammatory tumor associated macrophages are linked to progression and poor prognosis in cancer. This protocol is a guide to differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. Currently there is no established model for anti-inflammatory THP-1 polarization.This protocol uses adequate resting periods and a prolonged polarization process to create a distinct M2-like macrophage subtype. Tumor development, for example, in colon cancer and tissue remodeling, are controlled by anti-inflammatory macrophages. Among studies investigating their function, standardized creation of a distinct M2-like subtype ensures reproducible findings.Begin by setting a timer for 150 seconds. Remove the frozen vial containing the THP-1 cell line from the liquid nitrogen and thaw the vial immediately at 37 degrees Celsius in a clean water bath, starting the timer as soon as the vial is put into the water bath. Loosen the cap to release the pressure built up due to the thawing process, ensuring that the tube opening does not contact the water to avoid contamination.Thaw the cell suspension until an ice chip about four millimeters in size is left within the vial and proceed to the next step immediately. Transfer the liquid phase of the cell suspension to a 15 milliliter tube containing nine milliliters of warm growth media. Then transfer one milliliter of this warm medium cell suspension into the THP-1 vial and back to the 15 milliliter tube to melt the remaining ice chip, and flush the vial to ensure no cells are left behind.Mix the suspension gently by pipetting it up and down with a one milliliter pipette. Remove approximately 10 microliters of the sample to count the cells for viability using trypan blue. The cells with clear cytoplasm are viable, a blue cytoplasm indicates cell death.While counting, spin down the warm cell suspension at 200 times G for seven minutes at 37 degrees Celsius. Remove the supernatant completely and resuspend the cells in growth medium to achieve a cell density of 500, 000 cells per milliliter. Mix the suspension gently and transfer 22 milliliters into T75 cell culture flasks each.Store the flasks upright in an incubator at 37 degrees Celsius with a 5%carbon dioxide concentration and exchange the growth media every three to four days. Prepare the cell suspension to seed the cells at a density of 300, 000 per milliliter per well into 24-well cell culture plates. Mix the medium gently and a prepare aliquots of 26 milliliters in 50 milliliter tubes.Transfer one milliliter of the cell-containing medium into each well of a 24-well plate. Mix the media gently by pipetting it up and down between transfers. Keep the freshly prepared working solution of PMA on ice and add 100 nanograms of PMA per well.Incubate the plates in the incubator without any further treatment for 72 hours. After 72 hours, remove the growth medium and replace it with one milliliter of fresh growth medium, ensuring not to touch the bottom of the wells with pipette tips. Let the cells rest for another 96 hours in the incubator.Remove the growth medium and replace it with one milliliter of fresh growth medium. Add 20 nanograms of interleukin 4 and 20 nanograms of interleukin 13 per well. Then let the cells rest for 48 hours in the incubator.Repeat the whole process after 48 hours and let the cells rest for another 48 hours in the incubator. Remove the growth medium and replace it with one milliliter of fresh growth medium. Then let the cells rest for 48 hours in the incubator.Remove the warm cell medium from each well and replace it with a one milliliter mixture of ice cold PBS without calcium and magnesium and 5%FBS. Then immediately place the cell plate on ice for 45 minutes. After 45 minutes on ice, scrape off the cells using mini cell scrapers.Gently transfer the detached macrophages to cold PBS and 5%FBS in a 15 milliliter tube kept on ice. The macrophages derived from the polarization method show the expression of CD14, as well as CD11b markers. The monocyte and macrophage markers CD14 and CD11b were expressed in 70.9%and 74.7%of cells, respectively.Cells showed low surface levels of the M1-like marker CD80 in 0.2%of cells, and high surface levels of the M2-like marker CD206 in 62.6%of cells. As THP-1 cells in medium tend to sink to the bottom of a vial and are easy to activate through mechanical stress, gentle mixing and handling of the cells is important. M2-like macrophages are reproducibly created, and have been characterized using QR-TPCR, ELISA, and flow cytometry.This is the basis for investigating anti-inflammatory mechanisms in cancer development or tissue remodeling.

Research

JoVE Journal

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An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

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Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

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Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

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Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

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Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

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Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

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A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

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Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...